Capturing enzyme structure prior to reaction initiation: tropinone reductase-II-substrate complexes.
Capturing enzyme structure prior to reaction initiation: tropinone reductase-II-substrate complexes.
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DOI：
10.1021/bi0272712
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发表时间：
2003-04
期刊：
影响因子：
2.9
通讯作者：
A. Yamashita;M. Endo;T. Higashi;T. Nakatsu;Yasuyuki Yamada;J. Oda;H. Kato
中科院分区：
文献类型：
--
作者：
A. Yamashita;M. Endo;T. Higashi;T. Nakatsu;Yasuyuki Yamada;J. Oda;H. Kato
为了了解酶的催化机制，确定与各个反应步骤相对应的晶体结构是至关重要的。在这里，我们报道了反应开始前酶-底物复合体的两种晶体结构：托品酮还原酶-II(TR-II)-NADPH和从相同晶体中确定的TR-II-NADPH-托品酮复合体。两种动力学结晶学技术的结合，基片的连续流动和劳埃衍射测量，使我们能够在反应进行之前捕捉到过渡结构。本研究中的酶-底物复合体与我们先前研究中的酶-产物复合体的结构比较表明，底物之一托烷酮相对于产物旋转，从而使活性中心的空间组织有利于反应的进行。活性中心残基的侧链也会改变它们的构象，以保持底物或产物空间的互补性，并帮助旋转运动。
To understand the catalytic mechanism of an enzyme, it is crucial to determine the crystallographic structures corresponding to the individual reaction steps. Here, we report two crystal structures of enzyme-substrate complexes prior to reaction initiation: tropinone reductase-II (TR-II)-NADPH and TR-II-NADPH-tropinone complexes, determined from the identical crystals. A combination of two kinetic crystallographic techniques, a continuous flow of the substrates and Laue diffraction measurements, enabled us to capture the transit structures prior to the reaction proceeding. A structure comparison of the enzyme-substrate complex elucidated in this study with the enzyme-product complex in our previous study indicates that one of the substrates, tropinone, is rotated relative to the product so as to make the spatial organization in the active site favorable for the reaction to proceed. Side chains of the residues in the active site also alter their conformations to keep the complementarity of the space for the substrate or the product and to assist the rotational movement.